Age-related macular degeneration (AMD) is the leading cause of blindness in individuals over age 60. The efficacy of the new vascular endothelial growth factor (VEGF) inhibitors for treatment of age- related wet macular degeneration (AMD) is highly variable from patient to patient, and sustained responses are unpredictable. There is a great need to develop novel functional biomarkers to a) predict which patients will respond to anti-VEGF therapy b) determine who will need re-treatment and how often they should be treated, and c) identify candidate therapeutic targets in wet macular degeneration that is refractory to therapy. We propose the new hypothesis that phosphorylated forms of the activated VEGFR receptors and downstream or interconnected signaling proteins associated with neovascularization, vascular permeability, apoptosis, and inflammation, will be shed into the vitreous remodeling of the retina in wet macular degeneration. Our preliminary studies have strongly confirmed this hypothesis and provide the rationale for the Aims of this 2 year exploratory development project. Aim 1:Test the hypothesis that phosphorylated forms of protein receptors (e.g. VEGFR) and other signal pathway proteins are shed into the vitreous of patients with age-related wet macular degeneration. We will compare the levels of 50 validated phosphorylated receptor proteins and signal pathway analytes involved in neovascularization (e.g.VEGFR, PDGFR), and downstream pathways associated with vascular permeability, hypoxia, inflammation, prosurvival/apoptosis, and adhesion in disease versus control, before and after treatment. Aim 2: Evaluate if the protein markers of Aim 1 identified in the vitreous of wet macular degeneration patients, correlate with clinical response to therapy. Aim 3: Perfect novel phospho-protein affinity capture technology as a general method to discover and MS sequence additional members of this novel biomarker class of phosphorylated proteins in the vitreous that may constitute the next generation of functional biomarkers. The innovative components are a) a synergistic collaboration between George Mason University and the National Retinal Institute that maximizes opportunities for future translation of the findings to routine clinical use. b) A paradigm-shifting hypothesis that predicts the existence of a novel class of functionally relevant biomarkers in the vitreous, c) A unique clinical research study set comprising vitreous samples collected from 50 patients before and after 3 to 6 cycles of anti-VEGF intraocular therapy, d) Novel highly sensitive and precise technology to measure under CAP/CLIA guidelines the novel phospho- protein analyte panel in 20 microliters, and e) novel technology for affinity capture and enrichment of phosphoproteins in vitreous as a discovery tool for the future. The overall objective is to demonstrate the clinical feasibility and treatment associated relevance for a new class of vitreous biomarkers.Age-related macular degeneration (AMD) is the leading cause of blindness in individuals over age 60. This proposal will develop novel vitreous biomarkers to a) predict which patients will respond to anti-VEGF therapy b) determine who will need re-treatment and how often they should be treated, and c) identify candidate therapeutic targets in wet macular degeneration that is refractory to therapy.